Peptidoglycan Induces Cyclooxygenase-2 Expression in Macrophages by Activating the Neutral Sphingomyelinase-Ceramide Pathway

Abstract

The sphingomyelin signal transduction pathway is known to play a role in mediating the action of various cytokines. Herein, we examined the role of neutral sphingomyelinase (nSMase)/ ceramide in peptidoglycan (PGN)-induced NF-B activation and cyclooxygenase-2 (COX-2) expression in macrophages. PGN-induced COX-2 expression was attenuated by an nSMase inhibitor (3-O-methyl-sphingomyeline, 3-OMS) and ceramidase, but not by an acidic SMase inhibitor (imipramine). C2-ceramide, bacterial SMase (which mimics cellular SMase activity), and a ceramidase inhibitor (N-oleoyl-ethanolamine) individually had no effect on COX-2 expression; however, they markedly enhanced PGN-induced COX-2 expression. PGN activated nSMase, but not acidic SMase, resulting in increased ceramide generation. PGN-induced nSMase activation and ceramide formation were inhibited by 3-OMS, but not by imipramine. PGNinduced COX-2 expression was inhibited by a p38 MAPK inhibitor (SB 203580) and dominant negative mutants of MAPK kinase (MKK) 3, MKK6, and p38 MAPK␣. 3-OMS selectively inhibited PGN-induced p38 MAPK and MKK3/6 activation, but not JNK or ERK1/2. C2-ceramide, bacterial SMase, and N-oleoyl-ethanolamine all induced p38 MAPK or MKK3/6 activation. The PGN-mediated increases in B-luciferase activity were also inhibited by 3-OMS and the p38 MAPK␣DN, but not by imipramine. Furthermore, C2-ceramide caused an increase in B-luciferase activity. Our data demonstrate for the first time that PGN activates the nSMase/ceramide pathway to induce MKK3/ 6/p38 MAPK activation, which in turn initiates NF-B activation and ultimately induces COX-2 expression in macrophages. The nSMase/ceramide pathway is required but might not be sufficient for COX-2 expression induced by PGN.

Andrographolide Enhances Nuclear Factor-κB Subunit p65 Ser536 Dephosphorylation through Activation of Protein Phosphatase 2A in Vascular Smooth Muscle Cells

“…Previous studies have demonstrated that the formation of ceramide catalyzed by nSMase plays a critical role in PP2A activation (35,36). In agreement with these observations, we noted that 3-OMe-SM, a specific inhibitor of nSMase, inhibited andrographolide-induced ceramide formation and PP2A activation.…”

Platelet protease-activated receptor (PAR)4, but not PAR1, associated with neutral sphingomyelinase responsible for thrombin-stimulated ceramide-NF- B signaling in human platelets

“…20 Our findings also revealed that 3-OMS, an inhibitor of nSMase, attenuated platelet activation (i.e. NF-κB activation and p38 MAPK phosphorylation) stimulated by thrombin and PAR4-AP, but not by PAR1-AP or C2-ceramide, indicating that PAR4-nSMase/ceramide acted as an upstream regulator for platelet activation.…”

“…Our previous study had indicated that NF-κB activation can be regulated by mitogen-activated protein kinases (MAPKs) in smooth muscle cells. 20 Three…”

Endogenous Interleukin-1β in Neuropathic Rats Enhances Glutamate Release from the Primary Afferents in the Spinal Dorsal Horn through Coupling with Presynaptic N-Methyl-d-aspartic Acid Receptors*

“…We found that perfusion of 3-OMS (30 M) (52,55) into the recording chamber significantly reduced the first amplitude of EPSCs by 29.85 Ϯ 1.68% (n ϭ 6, p Ͻ 0.001) and increased the PPR from 0.64 Ϯ 0.01 to 0.75 Ϯ 0.02 (n ϭ 6, p Ͻ 0.01) (Fig. 8B).…”